Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus‐transduced NLFK cells

Abstract

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

Recovery of Recombinant Canine Distemper Virus That Expresses CPV-2a VP2: Uncovering the Mutation Profile of Recombinant Undergoing 50 Serial Passages In Vitro

“…In this study, the IFA showed that the rCDV-VP2-expressed VP2 was primarily localized in the nucleus, as evidenced by a green (Alexa Fluor ® 488)-blue (DAPI) merged morphology in Figure 1E (the first panel). This was in contrast with another conclusion in a previous report, which displayed that nuclear transport of CPV VP2 in the assembled form was hampered in the absence of other viral components (Gilbert et al, 2005). Yuan and Parrish (2001), nevertheless, showed that the VP2, albeit expressed alone, could accumulate in the nucleus of the A72 cell (Yuan and Parrish, 2001).…”

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

“…structure/function relationships at the molecular/cellular levels have been carried out. These include deletions of [33] and epitope insertions in the loops of the virion [34], N-terminal fusion of foreign antigens to VP1 [35,36], N-terminal insertion and fusion [17,19,37] or deletions of VP2 [33,38], and C-terminal fusions to VP2 [34,37,39]. …”

Baculoviral Vectors for Gene Delivery: A Review

“…Norden Laboratories feline kidney (NLFK) [93] [26]. This notion is supported by the finding that direct injection of nucleocapsids into cytoplasm does not affect the nuclear import, which implies that endosomal escape is not necessarily a critical step [27].…”

“…For example, the efficient baculovirus transduction of liver cells is exploited to deliver the genomes of hepatitis B and C viruses [89,90] into hepatoma cell lines, and is harnessed to evaluate the efficacy of antiviral compounds [91,92]. The low baculovirus cytotoxicity is tackled for the subcellular targeting of canine parvovirus capsid proteins [93] and viral membrane proteins [94] in baculovirustransduced cells, and more importantly, for the development of cell-based assays in the drug discovery setting (for review see [95][96][97]). The baculovirus display technology is utilized to construct eucaryotic epitope libraries (for review see [98,99]) and generate monoclonal antibodies [100,101].…”